Peptides

Abstract

Assays which comprise oligopeptides and which are useful for determining free PSA protease activity in vitro and in vivo are described. Such oligopeptides comprise amino acid sequences that are recognized and proteolytically cleaved by free prostrate specific antigen (PSA). Also described are assays useful in identifying inhibitors of free PSA.

What is claim is: 
     
       1. An oligopeptide that comprises a sequence of amino acids that is recognized and selectively proteolytically cleaved by free prostate specific antigen. 
     
     
       2. The oligopeptide according to claim 1 wherein the sequence of amino acids is a) AsnLysIleSerTyrGln|Ser (SEQ.ID.NO.: 13),   b) LysIleSerTyrGln|Ser (SEQ.ID.NO.: 14),   c) GlyGluAsnGlyValGlnLysAspValSerGlnXaaSerlleTyr|SerGlnThrGlu (SEQ.ID.NO.: 15), or   d) GlyLysGlyIleSerSerGlnTyr|SerAsnThrGluGluArgLeu (SEQ.ID.NO.: 2); wherein Xaa is any natural amino acid.     
     
     
       3. The oligopeptide according to claim 2 wherein the sequence of amino acids is a) AsnLysIleSerTyrGln|SerSer (SEQ.ID.NO.: 16),   b) AsnLysIleSerTyrGln|SerSerSer (SEQ.ID.NO.: 17),   c) AlaAsnLysIleSerTyrGln|SerSerSer (SEQ.ID.NO.: 18),   d) LysIleSerTyrGln|SerSerSerThrGlu (SEQ.ID.NO.: 19),   e) GlyGluAsnGlyValGlnLysAspValSerGlnArgSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 4), or   f) GlyGluAsnGlyValGlnLysAspValSerGlnSerSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 5).   
     
     
       4. The oligopeptide according to claim 2 wherein the amino acid sequence is a) AsnLysIleSerTyrGln|SerSerSerThr (SEQ.ID.NO.: 10),   b) AsnLysIleSerTyrGln|SerSerSerThrGlu (SEQ.ID.NO.: 3),   c) AlaAsnLysIleSerTyrGln|SerSerSerThrGlu (SEQ.ID.NO.: 11), or   d) GlyGluAsnGlyValGlnLysAspValSerGlnArgSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 4).   
     
     
       5. The oligopeptide according to claim 2 wherein the amino acid sequence is a) GlyArgLysAlaAsnLysIleSerTyrGln|SerSerSerThrGluGluArgArg LeuHisTyrGlyGluAsnGly (SEQ.ID.NO.: 6).   
     
     
       6. The oligopeptide according to claim 1 which is selected from: AsnArgIleSerTyrGln|Ser (SEQ.ID.NO.: 21),   AsnLysValSerTyrGln|Ser (SEQ.ID.NO.: 22),   AsnLysMetSerTyrGln|SerSer (SEQ.ID.NO.: 23),   AsnLysLeuSerTyrGln|SerSer (SEQ.ID.NO.: 24),   AsnLysIleThrTyrGln|SerSerSer (SEQ.ID.NO.: 25),   AsnLysIleSerPheGln|SerSerSer (SEQ.ID.NO.: 26),   AsnLysIleSerTrpGln|SerSerSerThr (SEQ.ID.NO.: 27),   AsnLysIleSerTyrAsn|SerSerSerThr (SEQ.ID.NO.: 28),   AsnLysIleSerTyrGln|ThrSerSerThr (SEQ.ID.NO.: 29),   AsnLysIleSerTyrGln|Ser (SEQ.ID.NO.: 30),   GlnLysIleSerTyrGln|SerSer (SEQ.ID.NO.: 31),   AsnArglleThrTyrGln|SerSerSer (SEQ.ID.NO.: 32),   AsnArgIleSerPheGln|SerSerSerThr (SEQ.ID.NO.: 33),   AsnArgIleSerTrpGln|SerSerSerThr (SEQ.ID.NO.: 34),   AsnArgIleSerTyrGln|ThrSerSerThr (SEQ.ID.NO.: 35),   AsnLysIIeThrTyrGln|ThrSerSerThr (SEQ.ID.NO.: 36),   AsnLysLeuSerTyrGln|ThrSerSerThr (SEQ.ID.NO.: 37),   GlnLysLeuSerTyrGln|SerSerSerThr (SEQ.ID.NO.: 38),   AsnArgLeuSerTyrGln|ThrSerSerThr (SEQ.ID.NO.: 39),   AsnLysValSerPheGln|SerSerSerThr (SEQ.ID.NO.: 40),   AsnArgValSerTrpGln|SerSerSerThr (SEQ.ID.NO.: 41),   GlnLysValSerTyrGln|SerSerSerThr (SEQ.ID.NO.: 42),   GlnLysIleSerTyrGln|ThrSerSerThr (SEQ.ID.NO.: 43), or   AsnLysIleSerTyrGln|SerSerSerThr (SEQ.ID.NO.: 44).   
     
     
       7. The oligopeptide according to claim 1 which is GlyGluGlnGlyValGlnLysAspValSerGlnSerSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 45),   GlyGluAsnGlyLeuGlnLysAspValSerGlnSerSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 46),   GlyGluAsnGlyValAsnLysAspValSerGlnSerSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 47),   GlyGluAsnGlyValGlnArgAspValSerGlnArgSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 48),   GlyGluAsnGlyValGlnLysAspValSerGlnLysSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 49),   GlyGluAsnGlyValGlnLysAspLeuSerGlnThrSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 50),   GlyGluAsnGlyValGlnLysAspValSerGlnSerSerIlePhe|SerGlnThrGlu (SEQ.ID.NO.: 51),   GlyGluAsnGlyValGlnLysAspMetSerGlnSerSerIleTyr|ThrGlnThrGlu (SEQ.ID.NO.: 52),   GlyGluAsnGlyValGlnLysAspValSerGlnArgSerIleTyr|ThrGlnThrGlu (SEQ.ID.NO.: 53),   GlyGluAsnGlyValGlnLysAspValSerGlnSerSerIleTyr|SerGlnSerGlu (SEQ.ID.NO.: 54),   GlyGluAsnGlyValGlnLysAspValSerGlnArgSerIleTyr|SerAsnThrGlu (SEQ.ID.NO.: 55);   GlyLysAlaIleSerSerGlnTyr|SerAsnThrGluGluArgLeu (SEQ.ID.NO.: 56),   GlyArgGlyIleSerSerGlnTyr|SerAsnThrGluGluArgLeu (SEQ.ID.NO.: 57),   GlyLysGlyIleThrSerGlnTyr|SerAsnThrGluGluArgLeu (SEQ.ID.NO.: 58),   GlyLysGlyIleSerThrGlnTyr|SerAsnThrGluGluArgLeu (SEQ.ID.NO.: 59),   GlyLysGlyIleSerSerAsnTyr|SerAsnThrGluGluArgLeu (SEQ.ID.NO.: 60),   AlaLysGlyIleSerSerGlnTyr|SerAsnThrGluGluArgLeu (SEQ.ID.NO.: 61),   GlyLysGlyIleSerSerGlnPhe|SerAsnThrGluGluArgLeu (SEQ.ID.NO.: 62),   GlyLysGlyIleSerSerGlnTyr|ThrAsnThrGluGluArgLeu (SEQ.ID.NO.: 63),   GlyLysGlyIleSerSerGlnTyr|SerAsnSerGluGluArgLeu (SEQ.ID.NO.: 64), or   GlyLysGlyIleSerSerGlnTyr|SerAsnThrAspGluArgLeu (SEQ.ID.NO.: 65).   
     
     
       8. An assay for determining proteolytic activity of free prostate specific antigen in a sample, comprising the steps of: (a), reacting a substrate, wherein the substrate is an oligopeptide that comprises an oligomer of from about 7 to about 100 amino acids residues which includes in its amino acid sequence a specific sequence of amino acids that is recognized and selectively proteolytically cleaved by free prostate specific antigen, with the sample to give an assay mixture; and   (b), detecting whether the substrate has been cleaved.   
     
     
       9. The assay according to claim 8 wherein the step of detecting whether the substrate has been cleaved comprises analyzing the assay mixture by high performance liquid chromatography. 
     
     
       10. The assay according to claim 8 wherein the oligopeptide comprises a sequence of amino acids that is selected from: a) AsnLysIleSerTyrGln|Ser (SEQ.ID.NO.: 13),   b) LysIleSerTyrGln|Ser (SEQ.ID.NO.: 14),   c) GlyGluAsnGlyValGlnLysAspValSerGlnXaaSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 15), and   d) GlyLysGlyIleSerSerGlnTyr|SerAsnThrGluGluArgLeu (SEQ.ID.NO.: 2): wherein Xaa is any natural amino acid.     
     
     
       11. The assay according to claim 8 wherein the assay further comprises the step (c): (c), detecting the amount of the substrate that has been cleaved at various times, the times which are selected from 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours and 20 hours.   
     
     
       12. An assay for identifying compounds which inhibit the proteolytic activity of prostate specific antigen, comprising: (a), reacting a substrate, wherein the substrate comprises a oligopeptide that comprises an oligomer of from about 7 to about 100 amino acids residues which includes in its amino acid sequence a specific sequence of amino acids that is recognized and selectively proteolytically cleaved by free prostate specific antigen, with free prostate specific antigen in the presence of a test substance to give an assay mixture; and   (b), detecting whether the substrate has been cleaved, in which the ability of the test substance to inhibit proteolytic activity of prostate specific antigen is indicated by a decrease in the cleavage of the substrate as compared to the cleavage of the substrate in the absence of the test substance.   
     
     
       13. The assay according to claim 12 wherein the oligopeptide comprises a sequence of amino acids that is selected from: a) AsnLysIleSerTyrGln|Ser (SEQ.ID.NO.: 13),   b) LysIleSerTyrGln|Ser (SEQ.ID.NO.: 14),   c) GlyGluAsnGlyValGlnLysAspValSerGlnXaaSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.: 15), and   d) GlyLysGlyIleSerSerGlnTyr|SerAsnThrGluGluArgLeu (SEQ.ID.NO.: 2); wherein Xaa is any natural amino acid.     
     
     
       14. The assay according to claim 13 wherein the step of detecting whether the substrate has been cleaved comprises analyzing the assay mixture by high performance liquid chromatography. 
     
     
       15. The assay according to claim 8 wherein the detecting of the cleavage of the substrate in step (b) is performed at various times, the times which are selected from 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours and 20 hours.